-
Journal of Virology Feb 1986Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol... (Comparative Study)
Comparative Study
Sequences in the human genome with homology to the murine mammary tumor virus (MMTV) pol gene were isolated from a human phage library. Ten clones with extensive pol homology were shown to define five separate loci. These loci share common sequences immediately adjacent to the pol-like segments and, in addition, contain a related repeat element which bounds this region. This organization is suggestive of a proviral structure. We estimate that the human genome contains 30 to 40 copies of these pol-related sequences. The pol region of one of the cloned segments (HM16) and the complete MMTV pol gene were sequenced and compared. The nucleotide homology between these pol sequences is 52% and is concentrated in the terminal regions. The MMTV pol gene contains a single long open reading frame encoding 899 amino acids and is demarcated from the partially overlapping putative gag gene by termination codons and a shift in translational reading frame. The pol sequence of HM16 is multiply terminated but does contain open reading frames which encode 370, 105, and 112 amino acid residues in separate reading frames. We deduced a composite pol protein sequence for HM16 by aligning it to the MMTV pol gene and then compared these sequences with other retroviral pol protein sequences. Conserved sequences occur in both the amino and carboxyl regions which lie within the polymerase and endonuclease domains of pol, respectively.
Topics: Amino Acid Sequence; Base Sequence; Genes; Genes, Viral; Humans; Mammary Tumor Virus, Mouse; RNA-Directed DNA Polymerase; Repetitive Sequences, Nucleic Acid; Sequence Homology, Nucleic Acid
PubMed: 2418214
DOI: 10.1128/JVI.57.2.422-432.1986 -
Viral diversity is an obligate consideration in CRISPR/Cas9 designs for targeting the HIV reservoir.BMC Biology Jul 2018RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative...
BACKGROUND
RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV.
RESULTS
We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses.
CONCLUSIONS
Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation.
Topics: CRISPR-Cas Systems; Gene Editing; Gene Products, gag; Genes, pol; Genetic Therapy; Genetic Variation; HIV Infections; HIV Long Terminal Repeat; HIV-1; Humans; RNA, Guide, CRISPR-Cas Systems; Virus Latency
PubMed: 29996827
DOI: 10.1186/s12915-018-0544-1 -
Journal of Virology Apr 1992Intracisternal A-particle (IAP) retrotransposons of rodents express gag and pol proteins for assembly of intracellular viruslike particles but lack an env gene. The...
Intracisternal A-particle (IAP) retrotransposons of rodents express gag and pol proteins for assembly of intracellular viruslike particles but lack an env gene. The recently described IAP-related family of retroviral elements contains a reading frame with close resemblance to retroviral env genes (IAPEs) (F. U. Reuss and H. C. Schaller, J. Virol. 65:5702-5709, 1991). I now report the analysis of cellular IAPE mRNAs and detection of IAPE env proteins. IAPE elements are transcribed in cell lines NH15-CA2 and AtT20. Four major transcripts of 4.2, 3.9, 2.8, and 1.3 kb are detected and characterized by probes specific for defined regions of the cloned IAPE-1 cDNA. The 2.8-kb mRNA is shown to lack gag and pol genes but comprises an env gene and U3 region, as expected for a subgenomic env mRNA. Polymerase chain reaction amplification and cloning of such mRNAs confirmed the absence of gag and pol genes 5' from the env gene and implicates env mRNA generation by a splicing event. A polyclonal anti-IAPE env antiserum, raised against a bacterial IAPE-env fusion protein, specifically detects N-glycosylated env proteins of 91 kDa or less in cell lines positive for IAPE mRNA. IAPE env proteins of different sizes represent independent translation products. After inhibition of N-glycosylation, env proteins in the size predicted from the env gene sequence or smaller are present. These results provide evidence that putative IAPE env proteins are synthesized in vivo. Envelope protein expression by an IAP-related retroviral element identifies IAPEs as a possible missing link between IAP retrotransposons and retroviruses.
Topics: 3T3 Cells; Animals; Base Sequence; Cell Line; DNA, Viral; Gene Products, env; Genes, Intracisternal A-Particle; Genes, gag; Genes, pol; Glycosylation; Mice; Molecular Sequence Data; Protein Biosynthesis; RNA Splicing; RNA, Transfer, Phe; Radioimmunoprecipitation Assay; Sequence Homology, Nucleic Acid; Transcription, Genetic; Tumor Cells, Cultured
PubMed: 1548748
DOI: 10.1128/JVI.66.4.1915-1923.1992 -
Scientific Data Jul 2018Accurate classification of HIV-1 group M lineages, henceforth referred to as subtyping, is essential for understanding global HIV-1 molecular epidemiology. Because most...
Accurate classification of HIV-1 group M lineages, henceforth referred to as subtyping, is essential for understanding global HIV-1 molecular epidemiology. Because most HIV-1 sequencing is done for genotypic resistance testing pol gene, we sought to develop a set of geographically-stratified pol sequences that represent HIV-1 group M sequence diversity. Representative pol sequences differ from representative complete genome sequences because not all CRFs have pol recombination points and because complete genome sequences may not faithfully reflect HIV-1 pol diversity. We developed a software pipeline that compiled 6,034 one-per-person complete HIV-1 pol sequences annotated by country and year belonging to 11 pure subtypes and 70 CRFs and selected a set of sequences whose average distance to the remaining sequences is minimized for each subtype/CRF and country to generate a Geographically-Stratified set of 716 Pol Subtype/CRF (GSPS) reference sequences. We provide extensive data on pol diversity within each subtype/CRF and country combination. The GSPS reference set will also be useful for HIV-1 pol subtyping.
Topics: Genes, pol; HIV Infections; HIV-1; Humans; Molecular Epidemiology; Recombination, Genetic
PubMed: 30063225
DOI: 10.1038/sdata.2018.148 -
Journal of Virology May 1989Sequence analysis of human T-cell leukemia proviral DNA revealed three open reading frames arranged at a -1 position relative to one another. On the basis of homology to...
Sequence analysis of human T-cell leukemia proviral DNA revealed three open reading frames arranged at a -1 position relative to one another. On the basis of homology to other retroviruses, these open reading frames were assigned to the gag, pro, and pol genes. To characterize the primary protein products of these genes and their modes of synthesis, a DNA clone of human T-cell leukemia virus type 2 was transcribed and translated in vitro. Analysis of the viral proteins revealed three polyproteins with molecular masses of 58, 75, and 112 kilodaltons at relative frequencies of 100:13:0.9, respectively. These proteins were mapped on the viral genome by both internal deletions and 3'-end truncations at gag, pro, and pol, respectively. The results indicate that translation of the pol gene requires two independent frameshift events, and the readthrough frequencies at the two frameshift sites appeared to be similar.
Topics: Chromosome Mapping; Gene Expression Regulation; Gene Products, gag; Genes, Viral; Human T-lymphotropic virus 2; Molecular Weight; Protein Biosynthesis; Protein Precursors; RNA, Viral; RNA-Directed DNA Polymerase; Restriction Mapping; Retroviridae Proteins
PubMed: 2467996
DOI: 10.1128/JVI.63.5.2400-2404.1989 -
Journal of Virology Jan 2002A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons... (Comparative Study)
Comparative Study
A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.
Topics: AIDS Vaccines; Amino Acid Sequence; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Count; Genes, pol; HIV-1; Humans; Immunization; Macaca mulatta; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Plasmids; Spleen; T-Lymphocytes, Cytotoxic; Time Factors; Vaccines, DNA; Vaccines, Synthetic
PubMed: 11739684
DOI: 10.1128/jvi.76.1.185-194.2002 -
Frontiers in Immunology 2021Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most...
Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the genes were defective at both the genome and transcript levels. We speculate that the defective PERV genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV genes as a source animal species for xenotransplantation.
Topics: Amino Acid Sequence; Animals; Cell Line; Cells, Cultured; China; Endogenous Retroviruses; Gene Expression Profiling; Gene Products, pol; Genes, pol; Genome; Genome, Viral; HEK293 Cells; Humans; Proviruses; Sequence Homology, Amino Acid; Swine; Swine, Miniature; Transcription, Genetic; Transplantation, Heterologous
PubMed: 35126361
DOI: 10.3389/fimmu.2021.797608 -
Veterinary Sciences Aug 2019The viral expression in vivo, in bovine leukemia virus (BLV)-infected cattle, is considered to be restricted to extremely low levels, and the mitosis of infected B...
The viral expression in vivo, in bovine leukemia virus (BLV)-infected cattle, is considered to be restricted to extremely low levels, and the mitosis of infected B lymphocytes is regarded as the main mode of virus persistence within the infected host. In this study, the presence of BLV RNA in whole blood from seven asymptomatic cows naturally infected with BLV during one year, including a complete milking cycle and two delivery time points, was investigated by nested-PCR using the oligonucleotides complementary to the tax and pol gene. BLV RNA was detected in four cows at different time points, especially in high blood proviral load cows and around delivery time. This study describes for the first time the detection of free BLV RNA in blood from BLV-infected asymptomatic cows. The results obtained suggest the occurrence of persistent low-level expression of the tax and pol genes that could be a result of viral reactivation, within the asymptomatic period. This finding may be important in the pathogenesis of BLV infection, associated with the delivery period.
PubMed: 31390719
DOI: 10.3390/vetsci6030066 -
Kidney International Mar 1999The kidney is considered to play an important etiologic role in salt-sensitive hypertension. The aim of the present study was to isolate genes whose expression differs... (Comparative Study)
Comparative Study
BACKGROUND
The kidney is considered to play an important etiologic role in salt-sensitive hypertension. The aim of the present study was to isolate genes whose expression differs between the kidneys of salt-hypertensive and control rats using an mRNA differential display method.
METHODS
Dahl salt-sensitive (DS) and control salt-resistant rats (DR) were fed a 0.3% or 8% NaCl diet. Renal RNA was amplified by RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and compared among DR 0.3%, DR 8%, DS 0.3%, and DS 8%. Gene expression and localization were examined by Northern blotting, RNase protection assay, and in situ hybridization. Full-length nucleotide sequence was determined by screening a DS rat kidney cDNA library.
RESULTS
We identified one differentially displayed clone, and its expression was greater in DS than DR, which was not affected by salt loading. The sequence was 90% homologous to the 3'-noncoding region of the nicotinic acetylcholine receptor alpha7 subunit gene. Its expression was kidney-specific, and was localized in the proximal tubules. The transcript level was markedly increased precedent to the development of hypertension. Its expression was also high in other salt-sensitive rats, and low in normotensive Sprague-Dawley and Wistar rats. The full-length cDNA contained elements homologous to the retroviral pol gene, a primer binding site sequence for reverse transcriptase, and long-terminal repeats.
CONCLUSION
These results demonstrated that the newly identified transcripts (REPT1) belong to a novel retrotransposon family, which showed unique strain-, age-, tissue-, and cell type-specific expression pattern.
Topics: Animals; Base Sequence; DNA Primers; DNA, Complementary; Genes, pol; Hypertension; In Situ Hybridization; Kidney Tubules, Proximal; Male; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Rats; Rats, Inbred Dahl; Receptors, Nicotinic; Retroelements; Sequence Homology, Nucleic Acid; Species Specificity; Transcription, Genetic
PubMed: 10027936
DOI: 10.1046/j.1523-1755.1999.055003995.x -
RNA (New York, N.Y.) Dec 2003Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and...
Because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express Gag and Pol. To assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of Caenorhabditis elegans, Drosophila melanogaster, Schizosaccharomyces pombe, Candida albicans, and Arabidopsis thaliana. In contrast to the retroviruses, which typically encode pol in the -1 frame relative to gag, nearly half of the retroelements surveyed encode a single gag-pol open reading frame. This was particularly true for the Ty1/copia group retroelements. Most animal Ty3/gypsy retroelements, on the other hand, encode gag and pol in separate reading frames, and likely express Pol through +1 or -1 frameshifting. Conserved sequences conforming to slippery sites that specify viral ribosomal frameshifting were identified among retroelements with pol in the -1 frame. None of the plant retroelements encoded pol in the -1 frame relative to gag; however, two closely related plant Ty3/gypsy elements encode pol in the +1 frame. Interestingly, a group of plant Ty1/copia retroelements encode pol either in a +1 frame relative to gag or in two nonoverlapping reading frames. These retroelements have a conserved stem-loop at the end of gag, and likely express pol either by a novel means of internal ribosomal entry or by a bypass mechanism.
Topics: Base Sequence; Codon, Terminator; DNA; Genes, gag; Genes, pol; Molecular Sequence Data; Open Reading Frames; Phylogeny; Protein Biosynthesis; Repetitive Sequences, Nucleic Acid; Retroelements; Sequence Homology, Nucleic Acid
PubMed: 14623998
DOI: 10.1261/rna.5105503